Directed homologous recombination for genome engineering inEscherichia coli

2007 ◽  
Vol 58 (Supplement 1) ◽  
pp. 1-10
Author(s):  
B. Csörgő ◽  
G. Pósfai
Keyword(s):  
Homologous Recombination ◽  
Genome Engineering ◽  
Inescherichia Coli

scholarly journals Blocking drug efflux mechanisms facilitate genome engineering process in hypercellulolytic fungus, Penicillium funiculosum NCIM1228

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Anmoldeep Randhawa ◽  
Nandita Pasari ◽  
Tulika Sinha ◽  
Mayank Gupta ◽  
Anju M. Nair ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Genome Engineering ◽  
Efflux Pump ◽  
Genetic Manipulation ◽  
Function Analysis ◽  
Drug Tolerance ◽  
Growth Media ◽  
Penicillium Funiculosum ◽  
Cellobiohydrolase I ◽  
Drug Tolerability

Abstract Background Penicillium funiculosum NCIM1228 is a non-model filamentous fungus that produces high-quality secretome for lignocellulosic biomass saccharification. Despite having desirable traits to be an industrial workhorse, P. funiculosum has been underestimated due to a lack of reliable genetic engineering tools. Tolerance towards common fungal antibiotics had been one of the major hindrances towards development of reliable transformation tools against the non-model fungi. In this study, we sought to understand the mechanism of drug tolerance of P. funiculosum and the provision to counter it. We then attempted to identify a robust method of transformation for genome engineering of this fungus. Results Penicillium funiculosum showed a high degree of drug tolerance towards hygromycin, zeocin and nourseothricin, thereby hindering their use as selectable markers to obtain recombinant transformants. Transcriptome analysis suggested a high level expression of efflux pumps belonging to ABC and MFS family, especially when complex carbon was used in growth media. Antibiotic selection medium was optimized using a combination of efflux pump inhibitors and suitable carbon source to prevent drug tolerability. Protoplast-mediated and Agrobacterium-mediated transformation were attempted for identifying efficiencies of linear and circular DNA in performing genetic manipulation. After finding Ti-plasmid-based Agrobacterium-mediated transformation more suitable for P. funiculosum, we improvised the system to achieve random and homologous recombination-based gene integration and deletion, respectively. We found single-copy random integration of the T-DNA cassette and could achieve 60% efficiency in homologous recombination-based gene deletions. A faster, plasmid-free, and protoplast-based CRISPR/Cas9 gene-editing system was also developed for P. funiculosum. To show its utility in P. funiculosum, we deleted the gene coding for the most abundant cellulase Cellobiohydrolase I (CBH1) using a pair of sgRNA directed towards both ends of cbh1 open reading frame. Functional analysis of ∆cbh1 strain revealed its essentiality for the cellulolytic trait of P. funiculosum secretome. Conclusions In this study, we addressed drug tolerability of P. funiculosum and developed an optimized toolkit for its genome modification. Hence, we set the foundation for gene function analysis and further genetic improvements of P. funiculosum using both traditional and advanced methods.

Increasing the homologous recombination efficiency of eukaryotic microorganisms for enhanced genome engineering

2019 ◽  
Vol 103 (11) ◽  
pp. 4313-4324 ◽  
Author(s):  
Ying Ding ◽  
Kai-Feng Wang ◽  
Wei-Jian Wang ◽  
Yi-Rong Ma ◽  
Tian-Qiong Shi ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Genome Engineering ◽  
Recombination Efficiency ◽  
Eukaryotic Microorganisms

scholarly journals Efficient genome engineering by targeted homologous recombination in mouse embryos using transcription activator-like effector nucleases

2014 ◽  
Vol 5 (1) ◽  
Author(s):  
Daniel Sommer ◽  
Annika E. Peters ◽  
Tristan Wirtz ◽  
Maren Mai ◽  
Justus Ackermann ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Genome Engineering ◽  
Mouse Embryos ◽  
Transcription Activator

scholarly journals Assembly of Radically Recoded E. coli Genome Segments

2016 ◽  
Author(s):  
Julie E. Norville ◽  
Cameron L. Gardner ◽  
Eduardo Aponte ◽  
Conor K. Camplisson ◽  
Alexandra Gonzales ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Genome Engineering ◽  
Next Generation ◽  
Selective Marker ◽  
E Coli ◽  
Cassette Exchange ◽  
Large Potential ◽  
Attachment Sites

AbstractThe large potential of radically recoded organisms (RROs) in medicine and industry depends on improved technologies for efficient assembly and testing of recoded genomes for biosafety and functionality. Here we describe a next generation platform for conjugative assembly genome engineering, termed CAGE 2.0, that enables the scarless integration of large synthetically recoded E. coli segments at isogenic and adjacent genomic loci. A stable tdk dual selective marker is employed to facilitate cyclical assembly and removal of attachment sites used for targeted segment delivery by sitespecific recombination. Bypassing the need for vector transformation harnesses the multi Mb capacity of CAGE, while minimizing artifacts associated with RecA-mediated homologous recombination. Our method expands the genome engineering toolkit for radical modification across many organisms and recombinase-mediated cassette exchange (RMCE).

Bacterial Recombineering: Genome Engineering via Phage-Based Homologous Recombination

2015 ◽  
Vol 4 (11) ◽  
pp. 1176-1185 ◽  
Author(s):  
Gur Pines ◽  
Emily F. Freed ◽  
James D. Winkler ◽  
Ryan T. Gill
Keyword(s):  
Homologous Recombination ◽  
Genome Engineering

scholarly journals Short homology-directed repair using optimized Cas9 in the pathogen Cryptococcus neoformans enables rapid gene deletion and tagging

2021 ◽  
Author(s):  
Manning Y. Huang ◽  
Meenakshi B. Joshi ◽  
Michael J Boucher ◽  
Sujin Lee ◽  
Liza C. Loza ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Cryptococcus Neoformans ◽  
Genome Engineering ◽  
Fluorescent Protein ◽  
Pcr Amplification ◽  
Sexual Cycle ◽  
Calmodulin Binding ◽  
Homology Directed Repair ◽  
Recombination Efficiency ◽  
Localization Signal

Cryptococcus neoformans, the most common cause of fungal meningitis, is a basidiomycete haploid budding yeast with a complete sexual cycle. Genome modification by homologous recombination is feasible using biolistic transformation and long homology arms, but the method is arduous and unreliable. Recently, multiple groups have reported the use of CRISPR-Cas9 as an alternative to biolistics, but long homology arms are still necessary, limiting the utility of this method. Since the S. pyogenes Cas9 derivatives used in prior studies were not optimized for expression in C. neoformans, we designed, synthesized, and tested a fully C. neoformans-optimized Cas9. We found that a Cas9 harboring only common C. neoformans codons and a consensus C. neoformans intron together with a TEF1 promoter and terminator and a nuclear localization signal (C. neoformans-optimized CAS9 or 'CnoCAS9') reliably enabled genome editing in the widely-used KN99α C. neoformans strain. Furthermore, editing was accomplished using donors harboring short (50 bp) homology arms attached to marker DNAs produced with synthetic oligonucleotides and PCR amplification. We also demonstrated that prior stable integration of CnoCAS9 further enhances both transformation and homologous recombination efficiency; importantly, this manipulation does not impact virulence in animals. We also implemented a universal tagging module harboring a codon-optimized fluorescent protein (mNeonGreen) and a tandem Calmodulin Binding Peptide-2X FLAG Tag that allows for both localization and purification studies of proteins for which the corresponding genes are modified by short homology-directed recombination. These tools enable short-homology genome engineering in C. neoformans.

scholarly journals Streamlined CRISPR genome engineering in wild-type bacteria using SIBR-Cas

2021 ◽  
Author(s):  
Constantinos Patinios ◽  
Sjoerd C A Creutzburg ◽  
Adini Q Arifah ◽  
Belén Adiego-Pérez ◽  
Evans A Gyimah ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Genome Engineering ◽  
Wild Type ◽  
Editing Event ◽  
Pseudomonas Putida Kt2440 ◽  
Knock Out ◽  
Counter Selection ◽  
Exogenous Expression ◽  
Self Splicing ◽  
Control Delays

Abstract CRISPR-Cas is a powerful tool for genome editing in bacteria. However, its efficacy is dependent on host factors (such as DNA repair pathways) and/or exogenous expression of recombinases. In this study, we mitigated these constraints by developing a simple and widely applicable genome engineering tool for bacteria which we termed SIBR-Cas (Self-splicing Intron-Based Riboswitch-Cas). SIBR-Cas was generated from a mutant library of the theophylline-dependent self-splicing T4 td intron that allows for tight and inducible control over CRISPR-Cas counter-selection. This control delays CRISPR-Cas counter-selection, granting more time for the editing event (e.g. by homologous recombination) to occur. Without the use of exogenous recombinases, SIBR-Cas was successfully applied to knock-out several genes in three wild-type bacteria species (Escherichia coli MG1655, Pseudomonas putida KT2440 and Flavobacterium IR1) with poor homologous recombination systems. Compared to other genome engineering tools, SIBR-Cas is simple, tightly regulated and widely applicable for most (non-model) bacteria. Furthermore, we propose that SIBR can have a wider application as a simple gene expression and gene regulation control mechanism for any gene or RNA of interest in bacteria.

scholarly journals SIBR-Cas enables host-independent and universal CRISPR genome engineering in bacteria

2021 ◽  
Author(s):  
Constantinos Patinios ◽  
Sjoerd Creutzburg ◽  
Adini Arifah ◽  
Belen Perez ◽  
Colin Ingham ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Genome Engineering ◽  
Control Mechanism ◽  
Host Factors ◽  
Mutant Library ◽  
Editing Event ◽  
Knock Out ◽  
Exogenous Expression ◽  
Self Splicing ◽  
Control Delays

CRISPR-Cas is a powerful tool for genome editing in bacteria. However, its efficacy is dependent on host factors (such as DNA repair pathways) and/or exogenous expression of recombinases. In this study, we mitigated these constraints by developing a simple and universal genome engineering tool for bacteria which we termed SIBR-Cas (Self-splicing Intron-Based Riboswitch-Cas). SIBR-Cas was generated from a mutant library of the theophylline-dependent self-splicing T4 td intron that allows for universal and inducible control over CRISPR-Cas counterselection. This control delays CRISPR-Cas counterselection, granting more time for the editing event (e.g., by homologous recombination) to occur. Without the use of exogenous recombinases, SIBR-Cas was successfully applied to knock-out several genes in three bacteria with poor homologous recombination systems. Compared to other genome engineering tools, SIBR-Cas is simple, tightly regulated and widely applicable for most (non-model) bacteria. Furthermore, we propose that SIBR can have a wider application as a universal gene expression and gene regulation control mechanism for any gene or RNA of interest in bacteria.

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